A fundamental role of insulin is to facilitate the uptake of glucose from the blood stream to the insulin-responsive tissues of fat and muscle. In this process, insulin acts mainly by stimulating the recruitment of the facilitative glucose transporter isoform GLUT4 to the plasma membrane. Abnormalities in this process underlie insulin-resistance and type 2 diabetes. Thus, deeper understanding of the underlying signaling mechanisms will provide important information for the pathogenesis and treatment of these diseases. Protein kinase D (PKD) is a newly identified serine/threonine kinase family that has been implicated in protein transport. We have characterized a novel isoform of PKD, PKDnu, at both biochemical and cellular levels. Our proposal is based on our preliminary findings that PKDnu is activated by protein kinase C (PKC), an essential enzyme that regulates insulin-stimulated GLUT4 trafficking. Furthermore, we show that PKDnu directly interacts with the vesicle-associated membrane protein 2 (VAMP2), a critical GLUT4 vesicle-localized protein that mediates membrane fusion and GLUT4 trafficking. Therefore, we propose to determine the role of PKDnu in insulin and phorbol ester regulated GLUT4 trafficking. We hypothesize that PKDnu in response to insulin and phorbol ester treatment, promotes the transport of GLUT4-VAMP2-containing vesicles to the plasma membrane via its interaction with VAMP2 in insulin-responsive cells. The long-term interest of the study is to determine how PKD regulates membrane trafficking and to identify novel molecular targets for the treatment of diseases with deregulated protein transport. The study will provide insights to the molecular signaling mechanisms that mediate insulin-regulated GLUT4 trafficking. We will address the following specific aims: Aim 1. To test the hypothesis that PKDnu is activated by phorbol esters and insulin through a PKC-dependent pathway in insulin-responsive cells. Aim 2. To test the hypothesis that VAMP2 is a downstream target of PKDnu in insulin-responsive cells. Aim 3. To test the hypothesis that the PKC/PKDnu/VAMP2 signaling pathway contributes to phorbol ester and insulin-regulated recruitment of GLUT4 to the plasma membrane and glucose uptake.